Can Surgery Be Avoided? Exclusive Antibiotic Treatment for Pelvic Actinomycosis.

Pelvic actinomycosis is an uncommon, slowly progressing granulomatous infection that has been associated with the presence of intrauterine devices. Due to its unspecific clinical and radiologic findings, it can mimic pelvic or intra-abdominal malignancy leading to mutilating surgery of high morbidity. Rarely, diagnosis is made preoperatively and in most cases surgical intervention is necessary. The patient in our case is a 42-year-old female with an IUD for 15 years diagnosed with pelvic actinomycosis. Patient was uniquely diagnosed preoperatively through paracentesis and treated conservatively with prolonged antibiotic therapy and without any type of surgical intervention. Follow-up at 1 year showed almost complete radiologic resolution of the inflammatory mass, nutritional recovery, and absence of symptoms. Pelvic actinomycosis can be successfully diagnosed and treated medically without surgical interventions.

Assessing cellular DNA damage from a helium plasma needle.

The aim of the present study is to determine the deoxyribonucleic acid (DNA) damage by cells exposed to atmospheric pressure non-thermal plasma (APNTP). Mouse leukocytes embedded in agarose were exposed to the plasma at two different distances from a helium plasma needle outlet and during three different exposure periods. Damage was assessed by the single cell gel electrophoresis assay. The results indicate that, at 0.1 cm from the plasma needle, the exposure caused complete DNA fragmentation determined by the presence of so called "clouds". Samples exposed at 0.5 cm from the slide sample surface presented damage proportional to the exposure periods in terms of tail intensity, tail moment and "clouds" frequency. Studies performed with alkaline single cell gel electrophoresis assay to determine DNA breaks and alkali-labile sites, indicates that DNA damage produced by exposure to APNTP was caused mainly by oxidative radicals, rather than by UV light which causes cyclobutane pyrimidine dimers. These results allow us to conclude that plasma needle induced DNA breaks in mice leukocytes proportionally to exposure time.

The in vivo induction of sister chromatid exchange by the demethylating agent 5-aza-2'-deoxycytidine.

Previously, we observed that the demethylating agent 5-azacytidine (azaC) induces a constant and limited low frequency of sister chromatid exchange (SCE), seemingly due to a limited number of SCE-prone sites whose expression is related to DNA demethylation. An alternative explanation for the low frequency of SCE induction may be its inefficient incorporation into DNA, as it has a higher incorporation into RNA. The aim of the present work is to determine if the frequency of SCE induction is increased after exposure to 5-aza-2'-deoxycytidine (azadC), a compound with the same mechanism of demethylation as azaC but more efficiently incorporated into DNA. Groups of mice were treated with 2.2, 4.4, 6.6 and 8.8 µmol azadC per kilogram body weight, and the SCE frequency, the mitotic index and the average generation time were determined after two cell division cycles. The dose-response data of SCE induction showed two components: (i) a dose-dependent increase between 0 and 4.4 µmol and (ii) almost a same level of two SCEs per cell at 4.4 and 8.8 µmol. Although azadC is incorporated more efficiently into DNA, as shown by a lower dose required for a maximal effect, the highest frequency of SCE induction is similar to that observed with azaC. These results indicate that the low incorporation of azaC into DNA seems not to be the factor that limits SCE induction, but the limited number of specific SCE-prone sites in demethylated DNA. Perhaps, there are a restricted number of sites prone to homologous recombination due to DNA demethylation.

Radiosensitization of murine normoblasts in vivo by bromodeoxyuridine to the genotoxicity and cytotoxicity of the bone-seeking radiopharmaceutical 153Sm-EDTMP.

Abstract To establish a basis for a possible strategy for bone marrow ablation or therapy, we examined the effect of bromodeoxyuridine (BrdU) incorporation into DNA on the genotoxic and cytotoxic effects of samarium-153 ethylenediaminetetramethylene phosphonate ((153)Sm-EDTMP) in normoblasts in vivo. Cytotoxicity and genotoxicity were established by time-response curves of polychromatic erythrocyte (PCE) and micronucleated polychromatic erythrocyte (MN-PCE) frequencies, respectively, in mouse peripheral blood samples. The group treated with (153)Sm-EDTMP showed a clear induction of MN-PCEs; however, the group treated with BrdU plus (153)Sm-EDTMP paradoxically showed only a slight increase with respect to untreated controls. Treatment with (53)Sm-EDTMP caused a small reduction in PCE frequency, but exposure to BrdU or to BrdU plus (53)Sm-EDTMP reduced the PCE frequency significantly from 32 h to the end of the experiment. The PCE frequencies in the BrdU plus (53)Sm-EDTMP group were significantly lower than in the BrdU control group at the final time and were much lower than the group treated with only (53)Sm-EDTMP, which returned to basal values. The results suggest the radioinduction of a lethal lesion in BrdU-substituted DNA that cannot be repaired easily and does not permit cell division and micronucleus formation.

Effect of O6-chloroethylguanine DNA lesions on the kinetics and mechanism of micronucleus induction in vivo.

The aim of this work was to determine the kinetics of micronucleus production because of an increase in O(6)-chloroethyl guanine (O6-ChlEt-G) DNA lesions in murine bone marrow cells in vivo. We increased the frequency of O6-ChlEt-G lesions by pretreatment with an inhibitor of O(6)-methylguanine-DNA methyltransferase (MGMT), O(6)-benzylguanine (O6BG), and subsequent treatment with bis-chloroethylnitrosourea (BCNU). The kinetics of micronucleated-polychromatic erythrocyte (MN-PCE) induction was established by scoring the frequency of MN-PCEs per 2000 PCEs in peripheral blood at 8-hr intervals from immediately prior to treatment to 72-hr post-treatment. We examined groups of five mice treated with (i) dimethylsulfoxide (DMSO), (ii) O6BG in DMSO, (iii) BCNU, or (iv) O6BG in DMSO plus BCNU. The data indicate that O6BG pretreatment causes: (i) ían increase in MN-PCEs induced by BCNU, (ii) a delay in the time of maximal MN-PCE induction produced by the different BCNU doses, and (iii) an increase in cytotoxicity. These data confirm that O6-ChlEt-G is a lesion involved in DNA break induction and in the subsequent production of micronuclei, and also that these lesions seem to be stoichiometrically reduced by MGMT. These data also show that induction of MN-PCEs by BCNU is delayed by pretreatment with O6BG for more than 6 hr, perhaps due to the time required for repair of crosslinks derived from O6-ChlEt-G and/or for DNA duplication, which is required for adduct transformation into crosslinks.

Prometaphase accumulation in murine bone marrow cells following in vivo treatment with alloxan.

The aim of this study was to determine the effect of alloxan, an inhibitor of N-acetylglucosaminyl transferase that acts during the G2/M transition, on the course of mitosis in murine bone marrow cells. Mitotic cells from animals treated with different doses of alloxan were analyzed for the frequency of prometaphasic and metaphasic chromosomes based on their morphology and length. The results indicate that alloxan treatment substantially increases the frequency of prometaphase chromosomes. This suggests that N-acetylglucosaminyl transferase is also involved in the G2/M transition in bone marrow cells. Alloxan treatment also provides a method for obtaining large chromosomes for the analysis of chromosome bands, FISH and sister-chromatid exchanges.

Mechanisms of DNA breaks induction in vivo by 5-azacytidine: paths of micronucleus induction by azaC.

The aim of the present study was to correlate the time-response curves of micronucleated polychromatic erythrocyte (MN-PCE) induction by 5-azacytidine (azaC) with the possible processes involved in DNA break production; this is based on the results previously published by other authors. The MN-PCE induction at two different doses of azaC was determined by sampling blood from the tails of mice before the acute treatment and over nine periods of 8 h each afterwards. Both doses caused two peaks of MN-PCE induction, one at 32 h and another at 48 h, approximately; a shoulder was detected that remained high from 56 h up to the end of the study (72 h). These results suggest that azaC induced DNA breaks and subsequently MN (micronucleus) by three different mechanisms, and in agreement with data in the literature, these could be successively the following: (i) during excision of the large adduct comprising the DNA methyl transferase covalently linked to DNA; (ii) failure of recombination repair or mismatch repair; and (iii) persistent chromosome fragility in G-C rich sites due to DNA demethylation and chromatin decondensation.

Mechanism of in vivo sister-chromatid exchange induction by 5-azacytidine.

The aim of the present study was to explore the in vivo mechanism of sister-chromatid exchange (SCE) induction by 5-azacytidine (5-azaC) in murine bone marrow cells. Experiments were performed to examine SCE induction in response to different doses of 5-azaC as well as several exposures. Additionally, we examined the persistence of SCE induction and the effect of bromodeoxiuridine (BrdU) incorporation. Sister-chromatid differentiation was obtained by injecting mice intraperitoneally with BrdU absorbed to activated charcoal. Before BrdU injection, different doses of 5-azaC were administered intraperitoneally either singly or in multiples. Colchicine in an aqueous solution was administered subcutaneously 22 h after BrdU injection. Two hours later, animals were sacrificed by cervical dislocation and both femurs were dissected. Bone marrow cells were processed to obtain chromosome preparations, which were stained by the fluorescence plus Giemsa method. Results indicate that 5-azaC caused SCE, albeit to a limited extent. In order to discern whether the limitation was due to cytotoxicity or to partial 5-azaC incorporation, we administered multiple sub-toxic doses of 5-azaC. This treatment increased 5-azaC incorporation and reduced cytotoxicity, but did not raise SCE frequency, indicating that the limitation was not due to either of the two factors mentioned above. SCE frequency induced by 5-azaC persisted for at least eight cell divisions, confirming that this agent had caused inhibition of DNA methyltransferase and subsequently the reduction of DNA re-methylation, which in turn induced the expression of a number of SCE-prone sites. Finally, SCE induction in response to 5-azaC was completely dependent on BrdU incorporation. The data allow us to conclude that 5-azaC causes SCE to a limited extent; limited SCE induction was not due to the direct effect of incorporation or cytotoxicity of 5-azaC, but rather the generation of a number of SCE-prone sites, the expression of which persists for at least eight cell divisions and is dependent on BrdU incorporation.

Repairability during G1 of lesions eliciting sister chromatid exchanges induced by methylmethanesulfonate or ethylmethanesulfonate in bromodeoxyuridine-substituted and unsubstituted DNA strands.

The repairability during G(1) of DNA lesions eliciting sister chromatid exchanges (SCE) induced by methylmethanesulfonate (MMS) and ethylmethanesulfonate (EMS) in BrdU-substituted and unsubstituted DNA strands was determined in murine salivary gland cells in vivo. The SCE frequency was determined after exposure to MMS or EMS during early and late G(1) in the first or second cell division. The inducibility and repairability of SCE-eliciting lesions during G(1) in BrdU-substituted and unsubstituted strands were estimated considering that in the first division induction occurs on the unsubstituted strand and during the second division in one unsubstituted and one BrdU- substituted DNA strand. The results indicate that DNA lesions induced by MMS are 50% repaired in both the BrdU-substituted and unsubstituted strands and those induced by EMS are 60% repaired in the unsubstituted strand but only 20% in the BrdU-substituted strand. The increase in sensitivity of the BrdU-substituted strand to SCE induction with respect to the unsubstituted strand was 155 and 45% for MMS and EMS, respectively. These results imply that SCE-inducing lesions produced by MMS and EMS are only partially repaired and that BrdU incorporation could sensitize DNA not only to the induction of lesions eliciting SCE, but also to the induction of non-repairable lesions.

Sister chromatid exchange induction and the course of DNA duplication, two mechanisms of sister chromatid exchange induction by ENU and the role of BrdU.

The aims of the present study were to establish the following: (i) the course of sister chromatid exchange (SCE) induction by ethylnitrosourea (ENU) in the first, second and third divisions as a function of the exposure time; (ii) the persistence of SCE-inducing lesions and the determination of whether or not they are always involved in SCE formation; (iii) the effect of bromodeoxyuridine (BrdU) incorporation on the induction and persistence of SCE. Three-way differential staining of sister chromatids in murine bone marrow cells in vivo was used in the present study. The results indicate the following: (i) SCE induction in each cell division depends on the course of DNA duplication, suggesting that SCE occurs at the replication fork; (ii) the cell population under study could be considered synchronous and had a cell cycle duration of nearly 9 h; (iii) in the second and third cell divisions ENU preferentially induced SCE in the cycle in which the exposure occurred; (iv) lesions induced by exposure to ENU did not cause SCE at the same site in subsequent divisions; (v) ENU was also capable of producing a long-lasting induction of SCE in BrdU-unsubstituted DNA; (vi) the sensitivity to SCE induction by the mutagen increases nearly proportionally to BrdU incorporation into DNA.

Effect of disulfiram on the genotoxic potential of acetaldehyde in mouse spermatogonial cells.

The initial purpose of the study was to determine the potential of acetaldehyde (Ace) to increase the rate of sister-chromatid exchanges (SCEs) in mouse spermatogonia. We tested four doses of Ace (from 0.4 to 400.0 mg/kg), including a negative and a positive control group (distilled water and cyclophosphamide, respectively). The results showed that all tested doses were SCE inducers. The highest tested dose increased the control level more than three times. Also, the cumulative frequencies of SCEs per cell were higher in the Ace-treated animals than in the control cells. Ace is transformed into acetate through the enzyme aldehyde dehydrogenase, a process that may be blocked by disulfiram (Dis) generating the accumulation of Ace. The second purpose of the study was to determine if the administration of Dis (150 mg/kg) could increase the SCE rate produced by non-genotoxic doses of Ace. (0.004 and 0.04 mg/kg). The animals treated with the two doses of Ace alone showed no increase in the frequency of SCEs; also, Dis by itself was not an SCE inducer. However, the groups of animals previously treated with Dis showed an increase of 31 and 60% with respect to the values obtained with the two doses of Ace alone. Furthermore, the cumulative frequencies of SCEs per cell were higher in the animals administered with both compounds together than in those treated with them separately. These results suggest the need to extend this type of study to other models.

Kinetics of micronucleus induction and cytotoxic activity of colchicine in murine erythroblast in vivo.

In previous studies, we inferred some pharmacokinetic and pharmacodynamic parameters of alkylating agents and antimetabolites by comparing their kinetics of micronucleated polychromatic erythrocyte (MN-PCE) induction with the one obtained after the exposure to gamma rays in peripheral blood of mice, assuming that radiation acts immediately because it does not require absorption and distribution in the organism. According to our earlier studies, the kinetics of MN-PCE induction depends mainly on the following: (i) the cytotoxic effects that in turn could affect the duration of cell division; (ii) the pharmacokinetics including the metabolic activation requirement; and (iii) the mechanism of MN induction. The aim of the present study was to analyze the kinetics of MN-PCE induction by an aneuploidogen that induces micronuclei by acting on the achromatic spindle. The kinetics of MN-PCE induction by colchicine, as well as the reduction in the PCE frequency over time was determined in peripheral blood of mice treated with different doses of the aneuploidogen. The genotoxic effect, established as the area beneath the curve (ABC) of MN-PCE versus time-response, indicates an almost directly proportional relationship with respect to dose. Similarly, the relationship between dose and cytotoxic effect determined as the ABC of PCE versus time was inversely proportional, suggesting a relationship between both endpoints and doses administered. However, the number of cells affected by these two phenomena indicates that cytotoxicity is not necessarily caused, or at least not only by genotoxicity. The analysis of the kinetics of MN-PCE induction after the treatment with non-cytotoxic dose of colchicine, indicates that the MN-PCE appear in the blood stream at almost the same time, as occurs after the exposure to gamma rays; in spite of the differences in the cell cycle stage in which they can cause micronucleus (MN). Perhaps the fact that cells are not synchronized does not permit one to observe some difference in the time they appear in the blood. These results suggest that colchicine acts rapidly after exposure. The elimination half-life of colchicine is 17h, suggesting that colchicne is disposable for long time. With high doses of colchicine the pharmacokinetic parameters increases substantially. These data imply that low doses of colchicine are slightly cytotoxic, and that under this circumstances colchicines arrives rapidly to hemopoyetic tissues and acts for several hours.

Differences in sensitivity of murine spermatogonia and somatic cells in vivo to sister-chromatid exchange induction by nitrosoureas.

Previously published data indicate that spermatogonia (SPG) are less sensitive to a sister-chromatid exchange (SCE) induction for different mutagens. In an earlier study, we have observed that bromodeoxyuridine (BrdU) substituted murine SPG are less sensitive to SCE induction by gamma ray in cells, than bone marrow (BM) and salivary gland (SG) cells in vivo. This was interpreted to mean that SPG are more efficient in DNA repair or are less prone to SCE induction. That the lower induction of SCE could be due to a reduced accessibility of mutagens to the SPG by virtue of a physiological barrier, was discarded by using gamma radiation. The aim of the present study was to establish whether or not there are differences in SCE induction by nitrosoureas among SPG, SG and BM cells with BrdU substituted or unsubstituted DNA. It was observed that SCE induction by methylnitrosourea (MNU) or by ethylnitrosourea (ENU) in SPG was, respectively, five and two times lower than in SG, and ten and three times lower than in BM. In SPG after BrdU incorporation, there was no increase in efficiency of SCE induction; in fact, there was even a slight decrease by exposure to MNU or ENU. BM and SG cells showed an increased efficiency in SCE induction after BrdU incorporation. This implies that SPG are also less sensitive to SCE induction by nitrosoureas, which cause a different kind of damage from previously assayed mutagens.

Assessment of radiation-induced DNA damage caused by the incorporation of 99mTc-radiopharmaceuticals in murine lymphocytes using single cell gel electrophoresis.

The DNA damage induced by the 99mTc-radiopharmaceuticals incorporation to the cell was determined by the single-cell gel electrophoresis in murine lymphocytes in vitro. The 99mTc-hexamethyl-propylene amine oxime (99mTc-HMPAO) and 99mTc-2, 5-dihydroxybenzoic acid (99mTc-gentisic acid) induced nearly 100% of cells with breaks and/or alkali labile sites, which is explained by the action of the Auger electrons produced by the decay of the 99mTc. These results agree with the doses of 1.6 and 1.0 Gy estimated by subcellular dosimetry for 99mTc-HMPAO that is incorporated in the cytoplasm, and the 99mTc-gentisic acid, which remains bonded to the cell membrane, respectively. The results imply that Auger electrons are able to cause important DNA damage, when the radionuclide is incorporated in the range of a few microns from the nuclei.

Sister-chromatid exchanges induced by disulfiram in bone marrow and spermatogonial cells of mice treated in vivo.

Disulfiram is a widely used drug to treat alcoholism due to its capacity to inhibit the metabolism of acetaldehyde; however, its genotoxic potential is not well known. Thus, the aim of this investigation was to determine whether the chemical may induce sister-chromatid exchanges (SCEs) in an in vivo study using mouse bone marrow and spermatogonial cells. We used doses of 200, 400 and 800 mg/kg body weight and compared the obtained data with the values determined in a negative control group as well as with a positive control group (cyclophosphamide, 50 mg/kg). The results in both systems indicated a weak genotoxic response by the chemical. In the case of bone marrow, a significant SCE level was achieved only with the high tested dose, but in spermatogonial cells the three doses tested showed a significant difference with respect to the negative control. No significant alterations in the mitotic index or in the cell proliferation kinetics were observed in somatic cells. Concerning the effect of cyclophosphamide, an increase in the level of SCEs was observed in both types of cells, reaching more than three times the values obtained in their respective control groups.

Relationship between the kinetics of micronuclei induction and the mechanism of chromosome break formation by methylnitrosourea in mice in vivo.

The kinetics of micronucleated polychromatic erythrocytes (MN-PCE) induction by methylnitrosourea (MNU) was determined in mice with the purpose of discerning whether or not the kinetics reflects the mechanism of chromosome break induction. A very long latency period (LP) was observed which is not compatible with an agent that does not require metabolic activation or incorporation to DNA for acting, but this is consistent with the mechanism demonstrated earlier that MNU causes chromosome breaks throughout the repair of mismatches induced by the alkylation of bases in a previous division. This is also supported by the presence of two rates of MN-PCE induction with respect to dose, which suggests that MN-PCE are induced by two mechanisms, an efficient one induced with the lower dose, and another less efficient one induced with higher doses. A similar behavior was observed in the curve of LP vs. dose, the lower dose causes 8 h of LP and higher doses increase LP but not proportionally to dose. The lower dose did not cause a reduction in the proportion of polychromatic erythrocytes, suggesting that this dose did not produce an important cytotoxic effect that could explain the long LP.

In vivo repair during G1 of DNA lesions eliciting sister chromatid exchanges induced by methylnitrosourea or ethylnitrosourea in BrdU substituted or unsubstituted DNA in murine salivary gland cells.

The difference in efficiency of methylnitrosourea (MNU) and ethylnitrosourea (ENU) to induce SCE in early or late G1 was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation during G1. The repair during G1 was determined by treating the cells in early or late G1. Treatment was in the first cycle (G1 before incorporation of 5-bromodeoxyuridine (BrdU)) or in G1 of the second cycle (after a single round of BrdU incorporation). It was observed that 50% of the lesions induced by MNU that elicit SCE are repaired during G1. BrdU incorporation into DNA increases the sensitivity of the cell to SCE induction by MNU nearly 40%; however under this circumstance a slightly lower SCE frequency was observed in the cells exposed to MNU at early G1, indicating that during G1 only few lesions are repaired. The ENU-induced DNA-lesions involved in SCE production are nearly 100% persistent along G1; besides, a slight but significantly higher SCE frequency was observed in cells exposed at early G1, suggesting the formation of SCE-inducing lesions during G1. BrdU incorporation to DNA sensitizes the cell to SCE induction by ENU, increasing the SCE frequency to nearly to a 40%, although these additional lesions involved in SCE induction seem to be susceptible to repair during G1.

Repair kinetics of gamma-ray induced DNA damage determined by the single cell gel electrophoresis assay in murine leukocytes in vivo.

The repair kinetics of the gamma rays induced DNA damage was determined in murine peripheral blood leukocytes in vivo by the comet assay. Mice were exposed to 1.0 Gy of gamma rays in a 137Cs source and samples of peripheral blood were taken from their tails at different times. The repair was evaluated per mice in separate experiments by measuring the proportion of cells with tail (comets) in each sample. An average of nearly 80% of comets was obtained at the initial time after the exposure; 2 min later the frequency decreased to 45% and continued diminishing to 22% at 15 min. This evidences the presence of a rapid repair mechanism. For a period of 25 to 40 min after exposure there was a slight but consistent increase of comets from 22 to 38% followed by a second reduction, which could be due to a late repair process that causes strand breaks and then joined them. In summary our results indicated that this system seems to be appropriate for the study of the repair capacity of cells following exposure to ionizing radiation.

Damage-repair kinetics and early adaptive response induced by gamma rays in murine leukocytes in vivo.

The kinetics of damage induction and repair at different doses, and the adaptive response induced by gamma ray exposure were determined in murine leukocytes in vivo. The adaptive response was determined after an adaptive dose of 0.01 Gy and a challenge dose of 1.0 Gy administered 60 min later. DNA damage was measured by the single cell gel electrophoresis. Results indicate there is an early and efficient repair process that acts even during the exposure to radiation, which is able to reduce 80% of damaged cells. Later, an increase in damaged cells occurs, which seems to represent the breaks induced during the repair of other kinds of lesions. This suggests that mouse cells are genetically adapted to repair this kind of damage. It was found that the adaptive pretreatment reduces the percentage of damaged cells caused by the challenge dose to one third, and diminishes the damage produced during the late repair. This indicates that the early adaptive response is caused by the induction of a process that protects DNA from damage induction, i.e., synthesis of substances that scavenge free radicals.

Effect of vitamin C or beta-carotene on SCE induction by gamma rays in radiosensitized murine bone marrow cells in vivo.

The in vivo effect of vitamin C or beta-carotene on sister chromatid exchange (SCE) radio-induction was determined in murine bone marrow cells sensitized by BrdU incorporation. Pre- or post-treatment with 100 mg/kg body wt vitamin C did not cause a significant reduction in SCE induced by the exposure to 0.63 Gy gamma-rays. Treatment with a double dose of vitamin C and with 0.45 Gy radiation did not cause a significant reduction in SCE frequency. However, due to the fact that vitamin C per se is capable of SCE induction, if an additive effect of radiation and vitamin C is considered, the expected frequency is higher than that observed. This implies that vitamin C could have a slight radioprotective activity. With regard to beta-carotene, it has been demonstrated that 50 mg/kg body wt causes a statistically significant increase per se, although pre- and post-treatment with the same dose has an additive effect on SCE frequency induced by 0.62 Gy radiation. This indicates that beta-carotene does not have radioprotective activity under the conditions used in the present study.